Field of the Invention
The present invention relates to the fields of diagnostic and therapeutic neurology. More specifically, the present invention relates to, inter alia, methods of diagnosis and prognosis in multiple sclerosis.
Description of the Related Art
Progression through each phase of the cell cycle is controlled by specific cyclin dependent kinases (CDK) and their interactions with cyclins and CDK inhibitors (CKI). The expression of each cyclin fluctuates throughout the cell cycle, and CKI are down-regulated in response to mitogenic stimulation. Cyclin dependent kinases are a family of serine/threonine protein kinases that are regulated by multiple mechanisms leading to their activation at specific points of the cell cycle. Mitosis is regulated by CDC2 when in complex with cyclin B. Deregulation of the cell cycle is well documented in cancer, and compounds with CDK inhibitory activity have recently entered clinical trials. The expression of CDC2, CDK2 and CDK4 proteins is higher in colon cancer cells than in normal mucosa. Higher levels of cyclins DI, D3, A and E were also found in primary colorectal carcinomas than in the adjacent normal areas. It is also documented that CDC2 kinase activity is increased in colon cancer tissue, but not in normal tissue. CDC2 is mostly present in colon cancer cells positive for phosphorylated Rb protein, and its overexpression is higher in focal carcinomas. The cyclin dependent pathway is, however, complicated and other factors are necessary for proper function and progression through the cell cycle.
One of these other factors, the Response Gene to Complement (RGC)-32 was first cloned in the rat by differential display (1), and subsequently from human brain library (2). Overexpression of RGC-32 is associated with an increase in DNA synthesis, thus leading to the hypothesis that RGC-32 is involved in activation of the cell cycle (1). Experimental evidence indicates that RGC-32 has an important role in cell cycle activation through regulation of CDC2 kinase (2). Overexpression of RGC-32 in human aortic smooth muscle cells (SMC) increased BrdU incorporation and the number of cells progressing into G2/M phase. RGC-32 appears to complex with CDC2/cyclin BI and increase the kinase activity of CDC2. This kinase-enhancing activity requires CDC2 phosphorylation of RGC-32 at Threonine 91. These findings identify RGC-32 as a substrate and regulator of CDC2.
Thus, the Response Gene to Complement (RGC)-32, acts primarily as a cell cycle regulator (1-2). RGC-32 overexpression leads to an increase in DNA synthesis and cell cycle progression from the G1/G0 to G2/M phase (2). Both of these responses can be abolished by transfecting the cells with RGC-32-specific siRNA (3). RGC-32 forms complexes with CDC2 and enhances CDC2 kinase activity (2). Thus, RGC-32 appears to be a previously unrecognized regulator of CDC2, a critical kinase involved in cell cycle activation.
Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system and is a common cause of persistent disability in young adults. In patients suffering from MS, the immune system destroys the myelin sheet of axons in the brain and the spinal chord, causing a variety of neurological pathologies. In the most common form of MS, Relapsing-Remitting, episodes of acute worsening of neurological function (exacerbations, attacks) are followed by partial or complete recovery periods (remissions) that are free of disease progression (stable). It has been reported that ninety percent of patients with MS initially present with a clinically isolated syndrome because of an inflammatory demyelinating lesion in the optic nerve, brain stem, or spinal cord. About thirty percent of those patients with a clinically isolated syndrome progress to clinically definite MS within 12 months of presentation. The subsequent progression of the disease can vary significantly from patient to patient. The progression can range from a benign course to a classic relapsing-remitting, chronic progressive, or rare fulminant course.
A method for diagnosing MS that facilitates early MS diagnosis and prediction of disease activity (Benign, Moderate and Malignant) would be valuable for both managing the disease and providing counsel for the patient. For example, patients diagnosed early with active course of MS could be offered disease modifying treatments that have recently been shown to be beneficial in early MS.
Current methods for assessment and tracking progress of MS are based on assessment and scoring of patients' function in attacks and accumulated disabilities during the attacks. One assessment used to assess MS is the Expanded Disability Status Scale (EDSS). However, EDSS score system measures the outcome and does not have predict for the progression of the disease. In addition, EDSS scoring can be variable because it is based on a subjective assessment of patient function. Methods for diagnosis can also include tracking brain lesions by Magnetic Resonance Imaging (MRI) or testing Cerebrospinal Fluid (CSF) for Oligo-Clonal Banding (OCB). MRI is a physical method for assessment of brain lesions and is used widely for MS diagnosis. However, it has only very long term predictive value. In addition, the correlation between MRI results and disease activity is poor. Thus, MRI can not be used for short term projections of disease activity or disease management.
Cerebrospinal puncture is an unpleasant invasive procedure that is not suitable for routine use or prognosis. In addition, both methods assess damage only after it has occurred; neither method can predict the onset of attacks or silent, sub-clinical lesions. A further disadvantage in testing for Oligo-Clonal Banding, e.g., in CSF and MRI as a way to diagnose MS is that a negative Oligo-Clonal Banding or MRI will not preclude the existence of MS.
Most patients with MS initially present with a clinically isolated syndrome (CIS). Despite the fact that MS will develop in up to 80% of these patients, the course of the disease is unpredictable at its onset. The disease may remain inactive for many years before the appearance of a second clinical relapse or new lesions on MRI confirm the diagnosis. Because currently available therapy is only partially effective and side effects are common, many neurologists are uncertain whether to treat all such patients with immunomodulators, or to wait until the diagnosis is confirmed by a second clinical event or the appearance of new MRI lesions.
There is a need for a simple serological assay that predicts whether patients with a CIS suggestive of MS or newly diagnosed relapsing remitting MS will have a highly active disease course and therefore require aggressive treatment, or whether they will follow a more benign course that enables such patients to postpone immunomodulatory therapy until necessary. This assay would be also useful in helping the diagnosis of MS. There is also a need for a method that uses objectively assessed markers for diagnosing MS and for predicting disease activity, the onset of attacks or silent lesions in patients suffering from MS.
Little is currently known about the potential role of RGC-32 in autoimmune disorders, including multiple sclerosis (MS). Several studies have demonstrated impaired apoptosis of T cells in multiple sclerosis patients (4-6). Furthermore, relapses may be associated with the persistent presence of myelin-activated T cells resulting from impaired T-cell apoptosis (4-6). T-cell apoptosis in both experimental allergic encephalomyelitis (EAE) and multiple sclerosis is regulated in part by the Fas-FasL system (6), and ex vivo studies have demonstrated an increased resistance of T cells to Fas-mediated apoptosis during multiple sclerosis relapses (7). In addition, FasL expression has been found to be low during relapses, consistent with the increased resistance of the T cells to apoptosis (8). FasL expression on T cells is regulated by multiple factors, including the CDC2/cyclin B1 complex (9). Since RGC-32 binds to CDC2/cyclin B1 complex and up-regulates its activity, it is possible that RGC-32 is involved in regulating T-cell survival by modulating the expression of FasL. Preliminary studies have shown that RGC-32 is expressed by CD3+ as well as CD4+ T cells from peripheral blood (PB) and in brain tissue from MS patients (10-11).
Thus, there is a continued need in the art for improved methods and therapies to diagnose and treat multiple sclerosis and autoimmune diseases. The present invention fulfills this long standing need and desire in the art.